12. A method of preserving red blood cells, comprising:providing red blood
cells and a composition for red blood cell preservation, wherein said
composition comprises at least one potassium sparing agent and an
additive-composition comprising saline, adenine and glucose;
andcontacting said red blood cells and said composition such that said
red blood cells and said additive composition form a suspension.

13. The method of claim 12, further comprising the step:cooling said
suspension.

14. The method of claim 13, further comprising the step:storing said
cooled suspension for at least 2 weeks.

15. The method of claim 12, wherein said potassium agent is selected from
the group consisting of spironolactone, eplereone, amiloride, and
triamterene.

16. The method of claim 12, wherein said additive-composition further
comprises at least one additive agent selected from the group consisting
of mannitol, citrate, phosphate, and dextrose.

17. The method of claim 12, further comprising at least one potassium
channel blocker agent.

18. The method of claim 17, wherein said potassium channel blocker agent
is selected from the group consisting of apamin, clotramazole, cetiedil,
charybdotoxin, TEA, and Ba++.

19. The method of claim 12, further comprising at least one nitric oxide
donor agent.

20. The method of claim 19, wherein said nitric oxide donor agent is
selected from the group consisting of nitroglycerin, nitroprusside,
nicorandil, sydnonimines agents, statin agents, 1-arginine agents, and
tetrahydrobiopterin.

21. The method of claim 12, further comprising at least one antioxidant
agent.

22. The method of claim 21, wherein said antioxidant agent is selected
from the group consisting of polyphenolic agents, ascordbic acid,
fluvastatin, selenium, and α-tocopherol.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]The present application is a Section 371 U.S. national stage of
pending International Patent Application No. PCT/US2008/051324,
International Filing Date Jan. 17, 2008, which claims the benefit of
expired Provisional Patent Application No. 60/881,273, filed Jan. 19,
2007, all of which are hereby incorporated by reference in their
entireties.

[0005]Improved methods and compositions for storing red blood cells for
extended periods of time are needed. In particular, improved methods and
compositions for preserving red blood cell function for extended periods
of time are needed.

[0007]In certain embodiments, the present invention provides a composition
for preserving red blood cells, wherein the composition comprises at
least one potassium sparing agent. In some embodiments, the potassium
agent includes, but is not limited to, spironolactone, eplerone,
amiloride, triamterene, and any mineralocorticoid receptor blocking
agent. In some embodiments, the composition comprises at least one
additive agent includes, but is not limited to, adenine, glucose, saline,
mannitol, citrate, phosphate, and dextrose. In some embodiments, the
composition comprises at least one potassium channel blocker agent. In
some embodiments, the potassium channel blocker agent includes, but is
not limited to, apamin, clotramazole, cetiedil, charybdotoxin, TEA, and
Ba++. In some embodiments, the composition comprises at least one
nitric oxide donor agent. In some embodiments, the nitric oxide donor
agent includes, but is not limited to, nitroglycerin, nitroprusside,
nicorandil, sydnonimines agents, statin agents, 1-arginine agents, and
tetrahydrobiopterin. In some embodiments, the composition comprises at
least one antioxidant agent. In some embodiments, the antioxidant agent
includes, but is not limited to, polyphenolic agents, ascordbic acid,
fluvastatin, selenium, and α-tocopherol. In some embodiments, the
composition is configured to prevent red blood cell storage lesions. In
some embodiments, the composition is configured to prevent diminished red
blood cell deformability.

[0008]The present invention is not limited to a particular method of
storing red blood cells with the improved additive compositions of the
present invention. In some embodiments, the methods include, but are not
limited to, the following steps: 1) obtaining a blood donation from a
subject; 2) separating the red blood cells from the plasma thereby
forming packed red blood cells (e.g., utilizing any standard laboratory
technique; centrifugation); 3) mixing the packed red blood cells with an
additive composition (e.g., compositions consisting of
adenine-glucose-saline, compositions consisting of
adenine-glucose-saline-mannitol, and compositions consisting of
adenine-glucose-saline-citrate-phosphate-dextrose) and at least one
additional additive agent so as to form a suspension of red blood cells,
wherein the at least one additional additive agent comprises one or more
of a spironolactone, eplerone, amiloride, triamterene, and any
mineralocorticoid receptor blocking agent, TEA, Ba++, clotrimazole,
cetiedil, nitroglycerin, nitroprusside, nicorandil, sydnonimines agents,
statin agents, 1-arginine agents, tetrahydrobiopterin, polyphenolic
agents, ascordbic acid, fluvastatin, selenium, α-tocopherol; 4)
cooling the suspension of red blood cells (e.g., to about 1 to 6°
C.; and 5) storing the cooled suspension of red blood cells (e.g.,
according to standard blood bank procedures) for a period of, for
example, 11 weeks or more.

[0012]The ability for red blood cells to deform depends on, for example,
nitric oxide and intracellular K content and their interrelationship. Red
blood cells have been shown to be capable of producing nitric oxide (see,
e.g., Kleinbongard, et al., 2006 Blood 107(7):2943-2951; incorporated
herein by reference in its entirety), and nitric oxide synthase
inhibitors have been shown to significantly reduce red blood cell
deformability, whereas nitric oxide donors and K channel blocker (TEA)
increased deformability (see, e.g., Bor-Kucukatay, et al., 2003 Am J
Physiol Heart Circ Physiol. 284(5):H1577-84; incorporated herein by
reference in its entirety). In addition, during early red blood cell
storage, significant amounts of K are reversed in the extracellular
medium, due to leakage from red blood cell and to the block of Na pump at
4° C. (see, e.g., Minetti et al., 2001 Biochim Biophys Acta.
1527(3):149-155; incorporated herein by reference in its entirety). Red
blood cell related potassium loss is a complex and cell age-dependent
process. In the absence of other osmotic process, K loss results in
overall cell shrinkage. After reinfusion, the alterations that cells have
suffered during storage begin to reverse. For example, lactate rapidly
diffuses out of the cells, the levels of 2,3-BPG and ATP begin to rise,
and cells recover, although at a slower rate, from the imbalance in
monovalent cations. However, red blood cells that suffered from excessive
swelling (e.g., red blood cells having storage lesions) are unable to
recover promptly. In addition, increased oxidative stress has been shown
to alter in erythrocyte rheology and facilitate potassium leak. For
example, hypochlorous acid, a powerful natural oxidant, has been shown to
produce, prior to hemolysis, changes in erythrocyte deformability
evidenced by ektacytometry, and an increase in K leak (see, e.g.,
Vissers, et al., 1994 Free Radic. Biol. Med. 16(6):703-712; incorporated
herein by reference in its entirety). For example, incubation of red
blood cells with an antioxidant component such as a polyphenol agent
prevents rheology alterations and hemolysis caused by hypochlorous acid
(see, e.g., Suwalsky, M., et al., 2006 Food Chem. Toxicol.
44(8):1393-1398; incorporated herein by reference in its entirety).
Consequently, there remains a need for improved additive solutions and
processes which will preserve red blood cell function during prolong
storage as well as increase survival after transfusion.

[0013]Accordingly, the present invention provides improved additive
compositions configured for storing red blood cells for extended periods
of time while preventing red blood cell storage lesions, retaining red
blood cell deformability, and increasing survival of the red blood cells
following transfusion. The improved additive compositions configured for
storing red blood cells for extended periods of time may be used at any
temperature range (e.g., 1 to 6° C.). The improved additive
compositions configured for storing red blood cells for extended periods
may be directly infused into any type of animal (e.g., mammals),
including but not limited to, dogs, cats, cows, humans, primates, etc.
The improved additive compositions configured for storing red blood cells
for extended periods may be used in any type of setting (e.g., military,
hospital, clinic).

[0014]The present invention is not limited to a particular method or
manner of improving upon standard additive compositions configured for
storing red blood cells. In some embodiments, the present invention
improves upon standard additive compositions by providing additional
agents to the additive compositions. The present invention is not limited
to a particular type or kind of standard additive composition configured
for storing red blood cells. Examples of standard additive-compositions
for storing red blood cells include, but are not limited to, compositions
consisting of adenine-glucose-saline, compositions consisting of
adenine-glucose-saline-mannitol, and compositions consisting of
adenine-glucose-saline-citrate-phosphate-dextrose (see, e.g., Hess, J.
R., 2006 Vox Sanguinis 91:13-19; U.S. Pat. Nos. 6,770,478, 6,527,957,
6,267,925, 5,789,151, 5,250,303, 5,248,506, 5,147,776, 4,812,310,
4,585,735; each incorporated herein by reference in their entireties).
The present invention is not limited to providing a particular type or
types of additional agent(s) to standard additive compositions configured
for storing red blood cells. In some embodiments, the additional agent(s)
is provided as, for example, a time-release pellet at multiple times
during the extended storage of the red blood cells. In some embodiments,
the additional agent(s) is provided at the beginning of the extended
storage of the red blood cells.

[0015]In some embodiments, at least one potassium sparing drug is provided
to standard additive compositions configured for storing red blood cells.
The present invention is not limited to a particular type or kind of
potassium sparing drug (e.g., spironolactone, eplerone, amiloride,
triamterene, and any mineralocorticoid receptor blocking agent). The
present invention is not limited to providing a particular amount of a
potassium sparing drug to a standard additive composition configured for
storing red blood cells. In some embodiments, the amount of potassium
sparing drug(s) provided is sufficient to prevent storage related
potassium leakage from the red blood cells. In some embodiments, the
amount of potassium sparing drug(s) provided is sufficient to permit
prolonged storage of red blood cells while preventing red blood cell
storage lesions, retaining red blood cell deformability, and increasing
survival of the red blood cells following transfusion.

[0018]In some embodiments, at least one antioxidant agent is provided to
standard additive compositions configured for storing red blood cells.
The present invention is not limited to a particular type or kind of
antioxidant agent (e.g., polyphenolic agents, ascordbic acid,
fluvastatin, selenium, α-tocopherol). The present invention is not
limited to provide a particular amount of a antioxidant agent to a
standard additive composition configured for storing red blood cells. In
some embodiments, the amount of antioxidant agent(s) provided is
sufficient to permit prolonged storage of red blood cells while
preventing red blood cell storage lesions, retaining red blood cell
deformability, and increasing survival of the red blood cells following
transfusion.

[0019]In some embodiments, any one or more combinations of a potassium
sparing drug, a potassium channel blocker agent, a nitric oxide donor
agent, and an antioxidant agent may be provided to standard additive
compositions configured for storing red blood cells for purposes of, for
example, prolonging storage of red blood cells while preventing red blood
cell storage lesions, retaining red blood cell deformability, and
increasing survival of the red blood cells following transfusion.

[0020]The present invention is not limited to a particular method of
storing red blood cells with the improved additive compositions of the
present invention. In some embodiments, the methods include, but are not
limited to, the following steps: 1) obtaining a blood donation from a
subject; 2) separating the red blood cells from the plasma thereby
forming packed red blood cells (e.g., utilizing any standard laboratory
technique; centrifugation); 3) mixing the packed red blood cells with a
standard additive composition (e.g., compositions consisting of
adenine-glucose-saline, compositions consisting of
adenine-glucose-saline-mannitol, and compositions consisting of
adenine-glucose-saline-citrate-phosphate-dextrose) and at least one
additional additive agent so as to form a suspension of red blood cells,
wherein the at least one additional additive agent is selected from the
group consisting of a spironolactone, eplreone, amiloride, triamterene,
TEA, Ba++, clotrimazole, cetiedil, nitroglycerin, nitroprusside,
nicorandil, sydnonimines agents, statin agents, 1-arginine agents,
tetrahydrobiopterin, polyphenolic agents, ascordbic acid, fluvastatin,
selenium, α-tocopherol; 4) cooling the suspension of red blood
cells (e.g., to about 1 to 6° C.; and 5) storing the cooled
suspension of red blood cells (e.g., according to standard blood bank
procedures) for a period of, for example, 11 weeks or more.

[0022]In some embodiments, the present invention blood samples containing
the compositions for red blood cell preservation of the present invention
are periodically tested so as to assure proper red blood cell quality.
Any type of method may be used to assess red blood cell quality during
the prolonged storage (e.g., use of cellulose strips (or other solid
surfaces) having antibodies (e.g., a predefined number of antibodies so
as to bind a known amount of sample) specific, for example, for
non-healthy red blood cells, or any type of attribute of a non-functional
red blood cell).

[0023]In some embodiments, the health of a subject who as received a blood
transfusion with blood having been stored with the compositions of the
present invention is periodically tested so as to assure proper red blood
cell quality. Any type of method may be used to assess red blood cell
quality during the prolonged storage (e.g., use of cellulose strips (or
other solid surfaces) having antibodies (e.g., a predefined number of
antibodies so as to bind a known amount of sample) specific, for example,
for non-healthy red blood cells, or any type of attribute of a
non-functional red blood cell).

[0024]All publications and patents mentioned in the above specification
are herein incorporated by reference. Although the invention has been
described in connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly limited to
such specific embodiments. Indeed, various modifications of the described
modes for carrying out the invention that are obvious to those skilled in
the relevant fields are intended to be within the scope of the following
claims.

Patent applications by Bertram Pitt, Ann Arbor, MI US

Patent applications by Maria Carolina Delgado, Doral, FL US

Patent applications by THE REGENTS OF THE UNIVERSITY OF MICHIGAN

Patent applications in class MAINTAINING BLOOD OR SPERM IN A PHYSIOLOGICALLY ACTIVE STATE OR COMPOSITIONS THEREOF OR THEREFOR OR METHODS OF IN VITRO BLOOD CELL SEPARATION OR TREATMENT

Patent applications in all subclasses MAINTAINING BLOOD OR SPERM IN A PHYSIOLOGICALLY ACTIVE STATE OR COMPOSITIONS THEREOF OR THEREFOR OR METHODS OF IN VITRO BLOOD CELL SEPARATION OR TREATMENT